This invention relates to the sequencing of fluorescence labeled DNA and the detecting of the DNA after irradiation by light from a laser.
In one class of techniques for sequencing DNA, identical strands of DNA are marked with fluorescent dye. The strands are marked by attaching specially synthesized fluorescent oligonucleotide primers to the strands of DNA.
The strands are separated into four aliquots. The strands in a given aliquot are either individually cleaved at or synthesized to any base belonging to only one of the four base types, which are adenine, guanine, cytosine and thymine (hereinafter A, G, C and T). The adenine-, guanine-, cytosine- and thymine- terminated strands are then electrophoresed for separation and the separated strands are irradiated by a laser and the emission detected. The rate of electrophoresis indicates the DNA sequence.
In a prior art sequencing technique of this class, the fluorescent dyes used as markers have their maximum emission spectra in the visible range, the DNA is subject to irradiation in the visible spectra, and visible spectra detectors and light sources are used. Generally photomultiplier tubes are used for detection.
The prior art techniques for DNA sequencing have several disadvantages such as: (1) because the dyes have their emission spectra in the visible light spectrum, the lasers used to excite the fluorescent markers and, under some circumstances, the detectors for the light tend to be expensive, such as for example, relatively large and expensive lasers are needed under some circumstances; and (2) they are relatively noisy.